Recombinant yellow fever virus and method of use thereof

ABSTRACT

The present invention provides recombinant yellow fever viruses (YFV), particularly live attenuated recombinant YFV, which comprise exogenous (i.e., non-YFV) nucleotide sequences which encode exogenous (i.e., non-YFV) amino acid sequences. These recombinant YFV viruses comprise an exogenous nucleic acid. Infection of a host cell with a recombinant YFV provides for expression of the exogenous nucleic acid in a host cell and production of an antigenic polypeptide encoded by the exogenous nucleic acid. Such recombinant YFV are useful in eliciting an immune response to the exogenous polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/177,449, filed Jan. 21, 2000, which application is incorporatedherein by reference.

GOVERNMENT RIGHTS

The United States Government may have certain rights in this applicationpursuant to U.S. Public Health Service grant A144343.

FIELD OF THE INVENTION

The invention relates generally to the field of recombinant viruses andinduction of specific immunity, specifically to induction oftumor-specific immunity.

BACKGROUND OF THE INVENTION

Tumor-specific cytotoxic T lymphocytes (CTLs) can prevent or eradicatetumors in a number of experimental systems and in patients with cancer(1-3). Clinical trials have demonstrated that 35% of patients withmelanoma treated with specific, tumor-reactive lymphocytes can achieveeither partial or complete tumor regression (4). The antigens recognizedby the T cells have, in some cases, been identified (5, 6). Althoughcancer cells may express tumor-associated antigens (TAAs), CTLs directedagainst TAAs are not efficiently elicited by the growing tumor and,therefore, the immune system fails to control tumor growth. Thus, itappears that tumor cells lack either immunogenicity and/or theappropriate co-stimulation required for CTL activation.

In contrast to tumor cells, viruses are strong inducers of cellularimmune responses. Thus, activation of the tumor-directed CTL response byvaccination with recombinant viruses expressing tumor-associatedantigens is a promising approach for the prevention and treatment ofmalignancies. Viral vaccine vectors that have been successfully used inexperimental cancer models include poxviruses, adenoviruses,picornaviruses and influenza viruses (7-10). However, because eachvaccine vector may present its own set of beneficial and adverseproperties, the search for new vectors continues to be an active area ofresearch. For example, clinical use of some vectors currently understudy may be limited by their record of safety, efficacy, potentialoncogenicity or induction of immunosuppression. In addition, preexistingimmunity against the vector could hinder the potency of treatment (8,11), and therefore alternative viral vectors are needed.

There is a need in the field for viral vectors that can be used toinduce immunity to a wide variety of antigens, including those presenton tumors. The present invention addresses this need, and providesrelated advantages as well.

SUMMARY OF THE INVENTION

The present invention provides recombinant yellow fever viruses (YFV),particularly live attenuated recombinant YFV, which comprise exogenous(i.e., non-YFV) nucleotide sequences which encode exogenous (i.e.,non-YFV) amino acid sequences. These recombinant YFV viruses comprise anexogenous nucleic acid. Infection of a host cell with a recombinant YFVprovides for expression of the exogenous nucleic acid in a host cell andproduction of an antigenic polypeptide encoded by the exogenous nucleicacid. Such recombinant YFV are useful in eliciting an immune response tothe exogenous polypeptide.

The recombinant live attenuated YFV express an exogenous nucleotidesequence which encodes an exogenous polypeptide, such as, but notlimited to, a polypeptide obtained from a pathogenic agent other thanYFV, a tumor antigen, and the like. These recombinant YFV are useful,when introduced into a mammalian subject, in eliciting an immuneresponse to the exogenous polypeptide in the subject. Thus, therecombinant YFV of the invention serve as immunization vehicles.

A wide variety of antigenic amino acid sequences can be incorporatedinto the YFV polyprotein, including those of microbial pathogens (e.g.,bacteria, parasites, viruses (other than YFV), fungi, and the like) andtumor-associated antigens. In general, following infection of a hostcell by the recombinant virus of the invention, the exogenouspolypeptide is proteolytically cleaved from the viral polyproteinprecursor into which it is incorporated. The exogenous polypeptide maythen be exported to the host cell surface, may be presented on the cellsurface as a peptide with a major histocompatibility antigen, may besecreted from the cell, or may remain in the cytoplasm of the cell. Inthe context of tumor immunotherapy, expression of a exogenouspolypeptide in a host elicits an immune response to the tumor, with theresult that the tumor cell mass and/or tumor cell number is reduced,development of a tumor is prevented or delayed, and/or the probabilitythat a tumor will develop is reduced.

The invention provides pharmaceutical compositions comprisingrecombinant YFV of the invention. Such compositions can be used, forexample, to reduce the severity of disease, reduce the risk of clinicaldisease, prevent the onset of a disease and/or to ameliorate the diseasevia recruitment of the host immune system.

The invention also provides methods of eliciting an immune response toan antigen in a mammalian subject. Such methods comprise administering arecombinant YFV of the invention to a mammalian subject so as to elicitan immune response to the exogenous polypeptide. The antigen can be ahost antigen or an antigen of a non-YFV pathogen.

The invention further provides methods of reducing or inhibiting tumorcell growth, and methods of reducing tumor cell mass and/or tumor cellnumbers. Such methods comprise administering a recombinant YFV of theinvention which comprises exogenous sequences encoding atumor-associated antigen (TAA)/epitope to a host bearing a tumor, suchthat the recombinant YFV enters a cell of the host and the exogenous TAApolypeptide is expressed on the surface of a host cell, is presented inthe context of an MHC molecule, or, alternatively, secreted from thehost cell. An immune response is elicited to a tumor which bears on itssurface an antigen which comprises the exogenous TAA polypeptide orwhich resembles the exogenous polypeptide sufficiently to elicit animmune response toward the tumor cell. The immune response to the tumorbearing the tumor-associated antigen on its surface is sufficient toreduce, inhibit, or eliminate the tumor.

The invention further provides methods of preventing tumor cell growth,and methods of reducing the probability that a tumor will form,comprising administering a recombinant YFV of the invention, whichcomprises a tumor-associated antigen/epitope-encoding nucleic acid, to ahost not bearing a tumor, such that the recombinant YFV enters a hostcell, the tumor-associated antigen is expressed on the host cell surfaceand/or presented in the context of an MHC molecule (e.g., MHC Class 1),and an immune response is elicited to the tumor-associated antigen. Theimmune response to the tumor-associated antigen is sufficient toprevent, or reduce the likelihood, of tumor development in the host.

A primary object of the invention is to provide a recombinant YFV thatprovides for production of an exogenous polypeptide that is suitable forinduction of an immune response to the polypeptide in a host followinginfection with the recombinant YFV. Such exogenous polypeptides include,but are not limited to, an antigen produced by the host (e.g., a tumorantigen), or an antigen from a non-YFV pathogen (e.g., a retroviralantigen).

An advantage of the invention is that the recombinant YFV of theinvention are live attenuated virus, which will continue to propagateuntil the intervention of the host's immune system.

Another advantage of the invention is that the YFV exhibits low toxicityin vivo.

Yet another advantage of the invention is that the YFV can express apolypeptide inside the host cell, and thus can provide for induction ofan immune response, particularly a cellular immune response thatinvolves, for example, antigen-specific cytotoxic T lymphocytes (CTLs).

Yet another advantage of the invention is that the immune responseelicited using the YFV of the invention is not limited to the infectedcells, as the immune system will also recognize cells ea ring theantigen expressed by the recombinant YFV. For example, the production ofa tumor antigen by the recombinant YFV can “break immune tolerance” totumor antigens, and induce an immune response against the tumoreffective to, for example, inhibit tumor growth.

These and other objects, advantages, and features of the invention willbecome apparent to those persons skilled in the art upon reading thedetails of the invention as more fully described below.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a yellow fever virus vector, YF-pOva,and strategy for expression of chicken ovalbumin. The top bar representsYF Vector genomic RNA. Boxes-below represent mature viral- proteins.White arrows indicate NS2B/NS3 cleavage sites, black triangles indicatecellular signal peptidase cleavage sites. Nucleotide sequences encodingthe ovalbumin K^(b) epitope SIINFEKL and flanking viral proteasecleavage were inserted at the N-terminus or at the junctions betweenC-prM and NS2B-NS3 (indicated by black arrows).

FIG. 2 depicts one-step growth curves of parental yellow fever 17Dstrain (YF-17D) and three YF virus vectors: YF-pOva-1 (open triangles),YF-pOva-2 (open circles), and YF-pOva-8 (closed squares). SW13 cellmonolayers were infected (MOI=5) with YF-17D, YF-pOva-1, YF-pOva-2, orYF-pOva-8. Virus production (pfu/ml) was determined at each time pointby plaque assay.

FIGS. 3A-3C depict in vivo activation of CD8+ T cells to Ova peptide.Tetrameric SIINFEKL/murine MHC class I molecule H-2 K^(b) complex boundto CD8⁺ splenocytes. Flow cytometry histograms illustrating tetramerbinding to gated CD8⁻ T lymphocytes of naive mice or mice infected withparental YF-17D or recombinant YF-pOva-8. Splenocytes were restimulatedby co-cultivation for five days with EL4 cells expressing SIINFEKL(EL4-SL8). The values indicate the percentage of CD8⁺ T lymphocytes thatbound the tetramer.

FIGS. 4A-D and 5A-D depict the results of immunization with YF vector,which induces protective and antigen-specific immunity to melanoma B16expressing Ova. C57BL/6 mice were immunized either twice every 2 weeksintravenously (i.v., FIGS. 4A and 5A), intraperitoneally (i.p., FIGS. 4Band 5B), subcutaneously (s.c., FIGS. 4C and 5C) or intramuscularly(i.m., FIGS. 4D and 5D) with YF-pOva-8 (3×10⁵ pfu/mouse) (closedsquares). As a control, mice were inoculated with either parental YF-17D(open squares) or saline (naive) (closed circles). Thirty days afterfirst immunization (day 0) animals were challenged with 5×10⁴ B16-Ova.FIGS. 4A-D: Local tumor growth. The size of the tumor was determinedevery five days and is plotted as the average tumor area +/− standarddeviation in square cm² vs. time post-challenge (days). FIGS. 5A-D:Survival is plotted as the percentage of surviving animals vs. time. Allexperiments included 10 mice per group and were repeated three times.

FIGS. 6A-F depict the results of inoculating mice having established B16 with YF-pOva-8. C57BL/6 mice were injected subcutaneously withmelanoma B16-Ova (5×10³ cells/mouse (FIGS. 6A-6C) or 1×10⁴ cells/mouse(FIGS. 6D-6F) at day 0 (tumor implantation). Animals received threesubcutaneous inoculations every three days of either PBS (naive) (closedcircles), parental 17D (YF-17D) (open squares) or YF-pOva-8 (4×10⁵pfu/mouse) (closed squares). Vaccines were administered at the day oftumor implantation (day 0, FIGS. 6A and 6D), five days (day 5, FIGS. 6Band 6E) or ten days (day 10, FIGS. 6C and 6F) after tumor inoculation.Mice were monitored for evidence of tumor growth by palpation andinspection twice a week.

FIG. 7 depicts the lungs of treated mice evaluated in a coded, blindedmanner for pulmonary metastases 30 days after the tumor inoculation. Thenumber of pulmonary metastases is shown for individual mice.

FIG. 8 depicts pictures of lungs treated with YF-pOva-8 or YF-17D at day30 after tumor inoculation.

FIGS. 9A and 9B depict survival plotted as the percentage of survivinganimals vs. time following implantation of 5×10⁴ cells (FIG. 9A) or1×10⁶ cells (FIG. 9B). All experiments included 10 mice per group.Naive: closed circles; YF-17D, open squares; YF-pOva-8, closed squares.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Before the present invention is described, it is to be understood thatthis invention is not limited to particular embodiments described, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting, since the scope ofthe present invention will be limited only by the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications mentioned herein areincorporated herein by reference to disclose and describe the methodsand/or materials in connection with which the publications are cited.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “arecombinant virus” includes a plurality of such viruses and reference to“the epitope” includes reference to one or more epitopes and equivalentsthereof known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing ate of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DEFINITIONS

The terms “polynucleotide” and “nucleic acid”, used interchangeablyherein, refer to a polymeric forms of nucleotides of any length, eitherribonucleotides or deoxynucleotides. Thus, this term includes, but isnot limited to, single-, double-, or multi-stranded DNA or RNA, genomicDNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine andpyrimidine bases or other natural, chemically or biochemically modified,non-natural, or derivatized nucleotide bases.

The terms “peptide,” “oligopeptide,” “polypeptide,” “polyprotein,” and“protein”, are used interchangeably herein, and refer to a polymericform of amino acids of any length, which can include coded and non-codedamino acids, chemically or biochemically modified or derivatized aminoacids, and polypeptides having modified peptide backbones.

“Recombinant,” as used herein, means that a particular DNA sequence isthe product of various combinations of cloning, restriction, and/orligation steps resulting in a construct having a structural codingsequence distinguishable from homologous sequences found in naturalsystems. Generally, DNA sequences encoding the structural codingsequence can be assembled from cDNA fragments and short oligonucleotidelinkers, or from a series of oligonucleotides, to provide a syntheticgene which is capable of being expressed in a recombinanttranscriptional unit. Such sequences can be provided in the form of anopen reading frame uninterrupted by internal nontranslated sequences, orintrons, which are typically present in eukaryotic genes. Genomic DNAcomprising the relevant sequences could also be used. Sequences ofnon-translated DNA may be present 5′ or 3′ from the open reading frame,where such sequences do not interfere with manipulation or expression ofthe coding regions. Thus, e.g., the term “recombinant” polynucleotide ornucleic acid refers to one which is not naturally occurring, or is madeby the artificial combination of two otherwise separated segments ofsequence. This artificial combination is often accomplished by eitherchemical synthesis means, or by the artificial manipulation of isolatedsegments of nucleic acids, e.g., by genetic engineering techniques. Suchis usually done to replace a codon with a redundant codon encoding thesame or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate adesired combination of functions.

By “construct” is meant a recombinant nucleic acid, generallyrecombinant DNA, that has been generated for the purpose of theexpression of a specific nucleotide sequence(s), or is to be used in theconstruction of other recombinant nucleotide sequences.

Similarly, a “recombinant polypeptide” or “recombinant polyprotein”refers to a polypeptide or polyprotein which is not naturally occurring,or is made by the artificial combination of two otherwise separatedsegments of amino acid sequences. This artificial combination may beaccomplished by standard techniques of recombinant DNA technology, suchas described above, i.e., a recombinant polypeptide or recombinantpolyprotein may be encoded by a recombinant polynucleotide. Thus, arecombinant polypeptide or recombinant polyprotein is an amino acidsequence encoded by all or a portion of a recombinant polynucleotide.

The term “immunologically active” or “immunogenic” refers to thecapability of the natural, recombinant, or synthetic peptide to induce aspecific humoral and/or cellular immune response in a mammal. As usedherein, “antigenic amino acid sequence,” “antigenic polypeptide,” or“antigenic peptide” means an amino acid sequence that, either alone orin association with an accessory molecule (e.g., a class I or class IImajor histocompatibility antigen molecule), can elicit an immuneresponse in a mammal.

As used herein, “an immune response” is meant to encompass cellularand/or humoral immune responses that are sufficient to inhibit orprevent infection, or prevent or inhibit onset of disease symptomscaused by a microbial organism, particularly a pathogenic microbialorganism, and/or to inhibit, reduce, or prevent proliferation of a tumorcell, and/or to reduce tumor cell numbers or tumor mass, and/or toreduce the likelihood that a tumor will form.

The term “tumor-associated antigen” is a term well understood in theart, and refers to surface molecules that are differentially expressedin tumor cells relative to non-cancerous cells of the same cell type. Asused herein, “tumor-associated antigen” includes not only completetumor-associated antigens, but also epitope-comprising portions(fragments) thereof. A tumor-associated antigen (TAA) may be one foundin nature, or may be a synthetic version of a TAA found in nature, ormay be a variant of a naturally-occurring TAA, e.g., a variant hasenhanced immunogenic properties.

The terms “antigen” and “epitope” are well understood in the art andrefer to the portion of a macromolecule which is specifically recognizedby a component of the immune system, e.g., an antibody or a T-cellantigen receptor. Epitopes are recognized by antibodies in solution,e.g., free from other molecules. Epitopes are recognized by T-cellantigen receptor when the epitope is associated with a class I or classII major histocompatibility complex molecule.

As used herein the term “isolated” is meant to describe a compound ofinterest (e.g., a virus, a peptide, etc.) that is in an environmentdifferent from that in which the compound naturally occurs. “Isolated”is meant to include compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified.

As used herein, the term “substantially purified” refers to a compoundthat is removed from its natural environment and is at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which it is naturally associated.

By “subject” or “individual” or “patient,” which terms are usedinterchangeably herein, is meant any subject, particularly a mammaliansubject, for whom diagnosis or therapy is desired, particularly humans.Other subjects may include cattle, dogs, cats, guinea pigs, rabbits,rats, mice, horses, and so on. Of particular interest are those subjectssusceptible to infection by yellow fever virus, e.g., subjects who cansupport YFV replication.

A “biological sample” encompasses a variety of sample types obtainedfrom an organism and can be used in a diagnostic or monitoring assay.The term encompasses blood and other liquid samples of biologicalorigin, solid tissue samples, such as a biopsy specimen or tissuecultures or cells derived therefrom and the progeny thereof. The termencompasses samples that have been manipulated in any way after theirprocurement, such as by treatment with reagents, solubilization, orenrichment for certain components. The term encompasses a clinicalsample, and also includes cells in cell culture, cell supernatants, celllysates, serum, plasma, biological fluids and tissue samples.

The terms “treatment,” “treating,” and the like are used herein togenerally refer to obtaining a desired pharmacologic or physiologiceffect. The effect may be prophylactic in terms of completely orpartially preventing a disease or symptom thereof and/or may betherapeutic in terms of a partial or complete cure for a disease and/oradverse effect attributable to the disease. “Treatment” as used hereincovers any treatment of a disease in a mammal, e.g., a human, andincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e., arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.In some embodiments, the invention is directed toward treating patientswith cancer. In these embodiments, “treatment” can include reducing orinhibiting tumor cell growth, eliminating a tumor, reducing metastasis,reducing or inhibiting tumor cell proliferation, reducing tumor cellmass, reducing tumor cell number, and reducing the probability that atumor will form.

The terms “cancer”, “neoplasm”, “tumor”, and “carcinoma”, are usedinterchangeably herein to refer to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterized by a significant loss of control of cell proliferation.Cancerous cells can be benign or malignant.

Recombinant Yellow Fever Virus The present invention providesrecombinant yellow fever viruses (YFV), particularly live attenuatedrecombinant YFV, which comprise exogenous ( i.e., non-YFV) nucleotidesequences which encode exogenous (i.e., non-YFV) amino acid sequences.Such recombinant YFV are useful in eliciting an immune response to theexogenous peptide. For simplicity, “exogenous” is used throughout asexemplary of such sequences, but is not intended to be limiting.

Yellow fever virus is an enveloped, positive-stranded RNA virus and amember of the flaviviridae genus. The genome is approximately 11 kb inlength and encodes a single polypeptide (16). This polypeptide precursoris proteolytically processed during and after translation, generatingthe functional proteins necessary for viral replication. Processing ismediated by cellular and viral proteases that recognize short specificamino acid sequences present at the junctions of the viral proteins. Theviral protease NS2B/NS3 mediates most of the cleavages of thenon-structural proteins in the cytosol of the infected cell (17-19).

The encoded exogenous polypeptide is expressed in the context of normalviral protein translation, preferably as a component of a recombinant orfusion precursor polypeptide. The recombinant polyprotein generallycomprises the exogenous polypeptide and viral polypeptide, andpreferably further comprises an artificial proteolytic recognition siteor sites. The proteolytic recognition site(s) can be positioned inrecombinant precursor polypeptide so that the precursor polyprotein isproteolytically processed by viral or cellular protease(s) so as torelease the free exogenous protein from the viral proteins.

The starting YFV, which is subsequently modified to include theexogenous sequences, is referred to as the “parent” YFV, which can be anative yellow fever virus (either pathogenic or, preferably,non-pathogenic), an attenuated yellow fever virus, a vaccine yellowfever virus strain or a recombinant yellow fever virus. Any of a varietyof strains of YFV can be used in generating recombinant YFV as describedherein. The nucleotide sequence of a number of YFV strains are availablein public databases, including, e.g., GenBank. An exemplary strain is“YFV 17D.” The nucleotide sequence of the YFV genome, as well as theamino acid sequence of the encoded viral polyprotein are found underGenBank Accession No. X03700, and are also described in Rice et al.((1985) Science 229:726-733), both of which are incorporated herein byreference in their entirety for the nucleotide and protein sequencesdisclosed therein. Production of yellow fever virions (viral particles)is well known in the art.

In general, an exogenous nucleic acid(s) inserted into the YFV genomecomprises a nucleotide sequence encoding an exogenous polypeptide (i.e.,non-YFV polypeptide) and at least one nucleotide sequence encoding aproteolytic cleavage site. The nucleotide sequence encoding theexogenous polypeptide may be flanked on either side by nucleotidesequences encoding proteolytic cleavage sites. Alternatively, thenucleotide sequence encoding the exogenous polypeptide may be flanked ononly one side by a nucleotide sequence encoding a proteolytic cleavagesite. In the latter case, the insertion site may be chosen such that,after insertion, the nucleotide sequence encoding the exogenouspolypeptide is flanked on either side by nucleotide sequences encodingproteolytic cleavage sites, one of which was present in the parent YFVgenome immediately adjacent the site of insertion. The exogenous nucleicacid sequence encoding the exogenous polypeptide and the nucleic acidsequence encoding the proteolytic cleavage sites can be positionedvarious sites within the YFV genome. As non-limiting examples, theexogenous nucleic acid sequence may be inserted at one or more of thefollowing locations: (1) the N-terminus of the viral polypeptide; (2)between viral proteins C and prM; (3) between viral proteins NS2A andNS2B; (4) between viral proteins NS2B and NS3; (5) between viralproteins NS3 and NS4A; and (5) NS4A and NS4B. The exogenous nucleic acidcan be inserted at other sites in the YFV genome. Preferably, insertionof the exogenous nucleic acid does not disrupt YFV protein function,and/or proteolytic processing of the viral polypeptide, and/or viralreplication. Whether viral replication is adversely affected can bedetermined using well-established techniques, including, but not limitedto, a plaque assay, and a one-step growth curve assay, as described inExample 1.

Unlike other vectors which will produce only one cycle of antigenexpression and/or which will stop expression without the intervention ofthe host immune system, the active recombinant virus of the inventionwill continue to propagate until the immune system is sufficientlyactivated to halt the infection. This produces a stronger immuneresponse against the exogenous antigenic peptide produced from the YFVas compared to the immune response that would be elicited usingconventional expression vectors (e.g., a viral replicon).

The recombinant YFV also exhibits low toxicity to a host upon infection.For example, YF-17D is a very safe and effective live viral vaccine,prepared from infected chicken embryos under standards developed by theWorld Health Organization. After vaccination, immunity is elicitedwithin 10 days in over 95% of vaccines (12) and neutralizing antibodiesdirected against the virus can be detected for more than 35 years (13).The vaccine safety record is outstanding: serious adverse reactions toYF-17D vaccine are extremely uncommon, and reversion to wild type isvirtually non-existent (14, 15).

Additional features may be incorporated into the design ofreplication-competent recombinant YFV viruses, such as polylinkersequences (e.g., EcoR1, Not1, BssH2, and Xho1) to facilitate the ease ofinsertion of desired foreign sequences into the recombinant vector.Also, variants, such as a poly-glycine tract, may be inserted adjacentto the inserted sequence so as to enhance the structural flexibility ofthe region and potentially increase the efficiency of proteolyticprocessing.

More than one nucleic acid sequence encoding an exogenous protein orpolypeptide to be produced can be included in the recombinantreplication-competent YFV virus which, as a result, produces thecorresponding number of exogenous proteins or polypeptides. The two ormore nucleic acid sequences can each encode a different product or canencode the same product (e.g., if enhanced production of a protein orpolypeptide is desired).

Insertion of exogenous nucleic acid can be accomplished by standardtechniques of molecular biology, such as described in numerous standardprotocol texts, including e.g., Current Protocols in Molecular Biology,(F. M. Ausubel, et al., Eds. 1987, and updates. Example 1 providesfurther guidance for how particular insertions were accomplished. Usingthese guidelines, any of a variety of exogenous nucleic acids can beinserted into the YFV genome:

The exogenous nucleic acid can be from about 12 to about 18, from about15 to about 24, from about 21 to about 30, from about 30 to about 60,from about 60 to about 90, from about 90 to about 120, from about 120 toabout 150, from about 150 to about 180, from about 180 to about 240,from about 240 to about 300, from about 300 to about 600, from about 600to about 1200, from about 1200 to about 1500, from about 1500 to about2100, from about 2100 to about 2400, from about 2400 to about 3000nucleotides in length.

Recombinant YFV as described herein can be used to induce an immuneresponse against an antigen in an individual and, e.g., provideprotection against challenge or infection by the exogenous pathogen(bacterial, viral, fungal, parasitic) in which the antigen occurs. Theyare, therefore, useful as vaccines to provide immune protection againstsuch pathogens.

Any DNA sequence which encodes a polypeptide or protein which, whenexpressed, produces protective immunity against, for example, apathogenic organism or against a condition or disorder associated withthe presence of or caused by an antigen, can be considered exogenousnucleic acids for the purpose of the present invention. Nucleic acidsequences encoding one or more exogenous polypeptides (e.g., antigens orepitopes) of interest can be included in a vaccine of the presentinvention. If more than one exogenous antigen or epitope of interest isencoded by the exogenous nucleic acid sequences, they can be antigens orepitopes of a single pathogen or antigens or epitopes from more than one(different). In a preferred embodiment, such an organism is a pathogenicmicroorganism. For example, such an exogenous epitope may be found onbacteria, parasites, viruses or fungi which are the causative agents ofdiseases or disorders. In addition, epitopes of allergens, sperm andcancer cells can be used. Thus, in some embodiments, where more than oneexogenous peptide is encoded by the exogenous nucleic acid sequences,the more than one exogenous peptide can be different epitopes found on asingle TAA.

The exogenous proteins in the vaccine formulations of the invention canalso comprise an epitope of an exogenous organism. When the exogenouspolypeptide is expressed in a vertebrate host, it elicits an immuneresponse that protects against a condition or disorder caused by orassociated with expression of or the presence in the host of, an antigencomprising the epitope. For example, in this embodiment of theinvention, exogenous proteins that comprise an epitope(s) or protein(s)of snake venom, bee venom, a hormone, sperm (for contraception), anallergy-inducing antigen or any other antigen to which an immuneresponse is desired, may be used. In another embodiment, atumor-specific antigen can be expressed as a recombinant exogenousprotein, for induction of a protective, or otherwise therapeutic, immuneresponse against cancer.

The gene sequences encoding the exogenous protein to be expressed by therecombinant virus according to the present invention, can be obtained bytechniques known in the art, including but not limited to, chemical orenzymatic synthesis, purification from genomic DNA of the microorganism,by purification or isolation from a cDNA encoding a TAA, by cDNAsynthesis from RNA of the microorganism, or by recombinant DNA methods(Maniatis et al., Molecular Cloning, A Laboratory Manual, 1982, ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.).

When they are used as vaccines, the recombinant YFV of the presentinvention are administered to an individual using known methods. Theywill generally be administered by the same routes by which conventional(presently-available) vaccines are administered and/or by routes whichmimic the route by which infection by the pathogen of interest occurs.They can be administered in a vaccine composition which includes, inaddition to the replication-competent recombinant virus, aphysiologically acceptable carrier. The composition may also include animmunostimulating agent or adjuvant, flavoring agent, or stabilizer.

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, intratumoral,subcutaneous, intradermal, intravenous, rectal, nasal, oral and otherparenteral routes of administration. Routes of administration may becombined, if desired, or adjusted depending upon the antigenic peptideor the disease. The vaccine composition can be administered in a singledose or in multiple doses, and may encompass administration of boosterdoses, to elicit and/or maintain immunity.

The recombinant YFV vaccine is administered in an “effective amount,”that is, an amount of recombinant YFV that is effective in a selectedroute of administration to elicit an immune response effective tofacilitate protection of the host against infection, or symptomsassociated with infection, by a pathogenic organism. In someembodiments, an “effective amount” of a recombinant YFV vaccine is anamount of recombinant YFV that is effective in a route of administrationto elicit an immune response effective to reduce or inhibit tumor cellgrowth, to reduce tumor cell mass or tumor cell numbers, or to reducethe likelihood that a tumor will form.

The amount of recombinant YFV in each vaccine dose is selected as anamount which induces an immunoprotective or other immunotherapeuticresponse without significant, adverse side effects generally associatedwith typical vaccines. Such amount will vary depending upon whichspecific immunogen is employed, whether or not the vaccine formulationcomprises an adjuvant, and a variety of host-dependent factors. Ingeneral, it is expected that each dose of vaccine will be sufficient togenerate, upon infection of host cells, about 1-1000 μg of protein,generally from about 1-200 μg, normally from about 10-100 μg. Aneffective dose of recombinant YFV nucleic acid-based vaccine willgenerally involve administration of from about 1-1000 μg of nucleicacid. An optimal amount for a particular vaccine can be ascertained bystandard studies involving observation of antibody titers and otherresponses in subjects. The levels of immunity provided by the vaccinecan be monitored to determine the need, if any, for boosters. Followingan assessment of antibody titers in the serum, optional boosterimmunizations may be desired. The immune response to the protein of thisinvention is enhanced by the use of adjuvant and or an immunostimulant.

Recombinant YFV viruses of the present can also be used as a system forproducing the exogenous polypeptide in host cells, such as mammalian,particularly human, cells or other cell types. The exogenous protein isthen isolated using standard methods.

In either application (i.e., immunization or tissue culture production)the exogenous nucleic acid sequence introduced into the YFV can be oneobtained from a source in which it occurs naturally, produced usinggenetic engineering methods or synthesized chemically or enzymatically.The exogenous nucleic acid sequence introduced into the virus can encodean entire antigen against which an immune response is desired orantigenic epitopes or portions, e.g., an immunogenic fragment of fromabout 4 to about 1000, from about 10 to about 500, from about 15 toabout 250, from about 20 to about 100, from about 25 to about 50 aminoacids. Inserted sequences are expected to present to the host immunesystem, antigenic structures defined both by primary sequence andstructural conformation.

Compositions comprising recombinant Yellow Fever Viruses of theinvention

The present invention further provides compositions, includingpharmaceutical compositions, comprising the recombinant YFV of theinvention.

Compositions comprising recombinant YFV of the invention may include abuffer, which is selected according to the desired use of therecombinant YFV, and may also include other substances appropriate tothe intended use. Those skilled in the art can readily select anappropriate buffer, a wide variety of which are known in the art,suitable for an intended use. In some instances, the composition cancomprise a pharmaceutically acceptable excipient, a variety of which areknown in the art and need not be discussed in detail herein.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, “Remington: The Scienceand Practice of Pharmacy”, 19th Ed.. (1995) Mack Publishing Co.

Pharmaceutical compositions can be prepared in various forms, such asgranules, tablets, pills, suppositories, capsules, suspensions, salves,lotions and the like. Pharmaceutical grade organic or inorganic carriersand/or diluents suitable for oral and topical use can be used to make upcompositions containing the therapeutically-active compounds. Diluentsknown to the art include aqueous media, vegetable and animal oils andfats. Stabilizing agents, wetting and emulsifying agents, salts forvarying the osmotic pressure or buffers for securing an adequate pHvalue, and skin penetration enhancers can be used as auxiliary agents.

When used as a vaccine, a recombinant YFV of the invention can beformulated in a variety of ways. In general, the vaccine of theinvention is formulated according to methods well known in the art usingsuitable pharmaceutical carrier(s) and/or vehicle(s). A suitable vehicleis sterile saline. Other aqueous and non-aqueous isotonic sterileinjection solutions and aqueous and non-aqueous sterile suspensionsknown to be pharmaceutically acceptable carriers and well known to thoseof skill in the art may be employed for this purpose.

Optionally, a vaccine composition of the invention may be formulated tocontain other components, including, e.g., adjuvants, stabilizers, pHadjusters, preservatives and the like. Such components are well known tothose of skill in the vaccine art.

Methods of using the recombinant Yellow Fever Viruses of the invention

The present invention provides methods for eliciting an immune responseto an antigen, comprising administering a recombinant YFV of theinvention to a mammalian subject, wherein the YFV enters a cell, theexogenous polypeptide is released by proteolytic cleavage, and an immuneresponse is elicited to the exogenous polypeptide.

In some embodiments, a polypeptide antigen expressed on a given tumorcell (e.g., a tumor associated antigen; “TAA”) is inserted into arecombinant YFV of the invention as described herein. Such recombinantYFV can be administered to an individual having, or suspected of having,a tumor. In some cases, such recombinant YFV can be administered to anindividual who does not have a tumor, but in whom protective immunity isdesired. As is often the case, the immune system does not mount animmune response effective to inhibit or suppress tumor growth, oreliminate a tumor altogether. Tumor-associated antigens are often poorlyimmunogenic; perhaps due to an active and ongoing immunosuppressionagainst them. Furthermore, cancer patients tend to be immunosuppressed,and only respond to certain T-dependent antigens. In these cases,introduction into the host of a recombinant YFV of the invention whichexpresses an exogenous peptide corresponding to an antigen expressed onthe tumor cell surface can elicit an immune response to the tumor in thehost. As shown in the Examples, a recombinant YFV comprising an insertencoding the sequence SIINFEKL (“Ova”) (SEQ ID NO.:1), which isrecognized specifically by CD8⁻ cytotoxic T cells (CTLs) when presentedon the surface of a cell together with a class I MHC molecule, wasintroduced into mice. When mice were subsequently challenged with atumor expressing Ova on its surface, a robust CTL response was mountedagainst the tumor.

Any of a variety of known tumor-associated antigens (TAA) can beinserted into YFV of the invention. The entire TAA may be, but need notbe, inserted. Instead, a portion of a TAA, e.g., an epitope, may beinserted. Tumor-associated antigens (or epitope-containing fragmentsthereof) which may be inserted into YFV include, but are not limited to,MAGE-2, MAGE-3, MUC-1, MUC-2, HER-2, high molecular weightmelanoma-associated antigen MAA, GD2, carcinoembryonic antigen (CEA),TAG-72, ovarian-associated antigens OV-TL3 and MOV 18, TUAN, alpha-fetoprotein (AFP), OFP, CA-125, CA-50, CA-19-9, renal tumor-associatedantigen G250, EGP-40 (also known as EPCAM), S100 (malignantmelanoma-associated antigen), p53, and p21ras.

Recombinant YFV comprising a TAA can be administered to an individual asdescribed above. Whether an immune response is elicited to a given tumorcan be determined by methods standard in the art, including, but notlimited to, assaying for the presence and/or amount of TAA-specificantibody in a biological sample derived from the individual, e.g., byenzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), andthe like; assaying for the presence and/or numbers of CTLs specific fora TAA; and the like. Examples of how to assay for the presence and/ornumbers of TAA-specific CTLs are found in the Examples section hereinbelow. Standard immunological protocols may be used, which can be foundin a variety of texts, including, e.g., Current Protocols in Immunology(J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach and W.Strober Eds. 1991).

In other embodiments, the exogenous (non-YFV) polypeptide is anantigenic polypeptide of a microbial pathogen. Such recombinant YFV canthen be administered to a host to prevent or treat infection by thepathogen, or to prevent or treat symptoms of such pathogenic infection.Of particular interest is the prevention or treatment of infection ordisease caused by microbial pathogens that, during the course ofinfection, are present intracellularly, e.g., viruses (e.g., HIV),bacteria (e.g., Shigella, Listeria, and the like), parasites (e.g.,malarial parasites (e.g., Falciparum), trypansomes, and the like), etc.Antigenic polypeptides of such microbial pathogens are well known in theart, and can be readily selected for use in the present recombinant YFVvaccine by the ordinarily skilled artisan.

Whether an immune response is effective can be determined by standardassays, including, but not limited to, measuring tumor cell mass,measuring numbers of tumor cells in an individual, and measuring tumorcell metastasis. Such assays are described in the Examples sectionherein below.

Using the methods and compositions described herein in connection withthe subject invention, an immunoprotective response against microbialinfection can be induced in any subject, human or non-human, susceptibleto infection by a microbial pathogen. Where the recombinant YFVcomprises an exogenous nucleic acid sequence encoding a TAA, the subjectmay be one that is known to have cancer, is suspected of having cancer,or does not have cancer, but in whom immunity to cancer is to beinduced.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric.

Example 1 Generating Recombinant Yellow Fever Virus

Materials and Methods

Plasmids and PCR fragments. Plasmids pYF5′3′ and pYFM5.2 which encodefor the complete YFV-17D sequence were kindly provided by Dr. CharlesRice. A full-length viral RNA can be generated from these plasmids by anin vitro ligation procedure (20). Briefly, we inserted a PCR generatedDNA fragment, encoding a 13 amino acid peptide from chicken ovalbuminflanked by BssHII and BstEII or ClaI and NdeI restriction enzyme sites,and followed by the viral peptidase (NS2B-NS3 complex) recognition site.We inserted the fragment at the following sites of the viral cDNA: theN-terminus of the viral polypeptide, or between proteins C-prM,NS2A-NS2B, NS2B-NS3, NS3-NS4A and NS4A-NS4B. To insert foreign sequenceswithin the structural region of the genome, PCR fragments containing theforeign sequences were cloned into plasmid p5′3′ at each differentlocation. To generate recombinants in the non-structural part of thegenome, 8 kb PCR fragments corresponding to the YFV-sequences in pYFM5.2and containing the inserts of interest were produced. These PCRfragments were used as substitutes for plasmid pYFM5.2 in the in vitroligation reaction described below.

Generation of Viruses from Plasmids. Production of a molecular clone ofYFV-17D was carried out following a similar procedure originallypublished by Rice and co-workers (20). Briefly, 5 μg of each plasmid (orthe corresponding sequences generated by PCR) were digested withrestriction enzymes Aat II and Apa I. After digestion, the plasmidcontaining the 5′ and 3′ ends of the viral genome and the fragment fromYFM5.2 corresponding to the middle region were each purified usinglow-melting agarose gel electrophoresis and ligated in equimolarconcentrations for 4 hours at 16° C. Ligase was inactivated byincubation for 20 minutes at 60° C. The ligated DNA was then digestedwith Xho I and used as the template for in vitro transcription by SP6RNA polymerase (Promega, Madison, Wis.) in the presence of m⁷GpppAmp(New England Biolabs, Beverly, Mass.). Without further purification,synthetic RNA was transfected into BHK-21 cells by electroporation (BTXelectro cell manipulator 600, San Diego, Calif.).

Viral Stocks. Cytopathic effect (CPE) was observed 3 to 5 days followingtransfection. Viruses were cloned from individual plaques produced inBHK-21 cells. To generate viral stocks, cloned viruses were propagatedin SW13 cells; supernatants of infected cells were cleared, aliquoted,titered and stored at −70° C.

Single-Step Growth Curves. Subconfluent SW13 cell monolayers were washedonce with PBS and infected at a multiplicity of infection of 5 pfu/cell.After a 2 hr incubation period at 37° C., the cells were washed twicewith PBS and then covered with L-15 medium supplemented with 10% FCS.Infected cell cultures were incubated at 37° C. for several days, and100 μl aliquots were recovered every six hours for a period of 6 days oruntil total CPE occurred. Titers were determined by plaque assay.

Analysis of Viral RNA by RT-PCR. After subsequent passages ofrecombinant viruses on SW13 cells, total cytoplasmic RNA was obtainedfrom infected cells following the method of (21). Reverse transcriptionwas carried out with Superscript (Gibco-BRL), using random hexamers andspecific primer (ATCGCGGACCGAGTGGTTTTGTGTTTGTCATCCAAAGGTCTGCTTATTCTTGAGC (SEQ ID NO.: 2)) and following the manufacturer's recommendedprotocol. After 1 hour incubation at 42° C., 2 μl of each reactionproduct was used as template in a PCR reaction using RtTh (Perkin-Elmer)and specific primers flanking the sequence to be studied(CAATGAGGCACTCGCAGCAGCTGG (SEQ ID NO.: 3) and TGCCCTAGCTCTGTGCGCTGCCCYF-pOva-8 (SEQ ID NO.: 4)). The amplified PCR product was analyzed byrestriction enzyme digestion and/or DNA sequencing.

Results

Generation of YFV 17 D recombinants expressing a chicken ovalbuminT-cell epitope. Foreign sequences (flanked by protease recognitionsites) are inserted in-frame at different positions within the YFpolyprotein precursor. In this way, the viral protease recognizes andcleaves the flanking proteolytic sites, freeing the exogenous antigenicsequences from the rest of the YF polyprotein, and all of the YFproteins are produced correctly and viral replication proceeds normally(FIG. 1). At several positions of the viral genome, we introducedsequences encoding a chicken ovalbumin CTL epitope followed by an eightamino acid cleavage site for the viral protease NS2B/NS3 (FIG. 1). Theinserted fragment encoded the amino acid sequence SIINFEKL (SEQ ID NO.:1), an epitope restricted to the murine MHC class I molecule H-2 K^(b)(25).

Recombinant live yellow fever viruses were recovered by transfection ofBHK cells with in vitro synthesized RNA. Insertion of exogenoussequences at three sites in the genome yielded viable recombinantviruses: the amino terminus, C-prM, and NS2B-NS3 junctions (theresulting viruses were named YF-pOva-1, YF-pOva-2 and YF-pOva-8,respectively). In contrast, insertion at the NS2A-NS2B, NS3-NS4A,NS4A-NS4B junctions abolished viral replication (data not shown). Theviruses were isolated from individual plaques and viral stocks weregenerated by two sequential passages in SW13 cells.

Plaque assays and one-step growth curves showed that recombinantsYF-pOva-1, YF-pOva-2 and YF-pOva-8 replicate at rates remarkably similarto the parental 17D strain (FIG. 2). Recombinant YF-pOva-8 replicatedwith identical kinetic to 17D and achieved nearly equivalent titers.Recombinant YF-pOva-1 and YF-pOva-2 replicated slower than the parental17D, exhibiting a lag in replication, and by three days post-infectionachieved only 10-20% the titer of 17D (FIG. 2). In view of the betterreplication properties of YF-pOva-8, this recombinant was used in allsubsequent studies to further characterize the immunogenicity of yellowfever vectors.

Genetic stability problems have been reported with some positive strandRNA virus vectors (26, 27). Therefore we confirmed the presence of theinserted sequence by RT-PCR and a diagnostic restriction enzyme digest.The original genetic structure of YF-pOva-8 was retained after sixpassages in BHK-21 cells. Thus, foreign peptides can be introducedbetween the NS2B and NS3 coding sequences of the yellow fever genomewithout major compromise of viral replication.

Example 2 Cells Infected with YF-pOva-8 Present the Ova-peptide SIINFEKLin a MHC Class I-restricted Manner

Materials and Methods

Cell lines. In addition to BHK-21 and SW13 cells already described, thefollowing cell lines were used in the antigen presentation assay or inthe tumor rejection challenge: B3Z (T-cell hybridoma), EL-4 (thymoma)and EL4-SL8 cells: B3Z cells and EL4-SL8 were kindly provided by NilabhShastri, University of California, Berkeley. EL4-SL8 cells stablyexpress and present the Ova CTL epitope (SIINFEKL (SEQ ID NO.: 1)). B3Zis a murine T cell hybridoma specific for K^(b) +SIINFEKL (SEQ IDNO.:1), which is transfected with the LacZ-reporter gene under thetranscriptional control of the IL-2 enhancer element. Thus, B3Z cellactivation can be easily detected by the expression of β-galactosidase(22). The C57BL/6-derived melanoma B16F0 and B16-OVA were a kind giftfrom Kenneth Rock, University of Massachusetts. B16-Ova (Mo5.20.10) is acell line that stably expresses ovalbumin constructed by transfection ofB16F0 with plasmid pAc-neo-Ova (1).

Antigen Presentation Assay. EL4 cells were mock-infected or infectedwith YF-17D and recombinant viruses at a MOI of 10 pfu/cell. After a 48hours incubation period at 37° C., the infected cells or the same numberof B16-Ova control cells were co-cultured with 5×10⁴ B3Z cells for 16hrs at 37° C. To determine the expression of β-galactosidase, cultureswere washed with PBS and then fixed with 1% formaldehyde/0.2%glutaraldehyde for 5 min at 4° C. Cells were washed again and incubatedwith a solution consisting of I mg/ml X-Gal, 5 mM potassiumferrocyanide, 5 mM potassium ferricyanide, and 2 mM MgCl₂ in PBS. Theywere then incubated overnight at 37° C. and examined microscopically forthe presence of β-galactosidase activity (blue cells).

Results

Cells infected with YF-pOva-8 present the Ova-peptide SIINFEKL in a MHCclass I-restricted manner. To determine whether cells infected withrecombinant yellow fever viruses are able to both express and presentantigens in an MHC class I-restricted manner, we infected EL4 cells withYF-pOva-8 and co-cultivated the infected cells with a T-cell hybridomathat recognizes SIINFEKL presented in the context of K^(b) MHC class Imolecules. The antigen is initially expressed in the cytoplasm of theinfected cell, but it should be transported and presented on the surfaceof the cell through the MHC class I pathway. We used the T-cellhybridoma B3Z, carrying a lacZ reporter gene under the transcriptionalcontrol of the interleukin-2 enhancer element NF-AT (22). Thus,TCR-specific stimulation of B3Z cells can be measured by the productionof β-galactosidase activity.

EL4 cells infected with YF-pOva-8 activated 10% of the hybridoma T-cellsafter 12-16 hours of co-cultivation. In contrast, co-cultivation withuninfected EL4 cells or the hybridoma B3Z by itself did not produceβ-galactosidase activity. Co-cultivation with a cell line that stablyexpresses ovalbumin strongly induced β-galactosidase production. Theseresults demonstrated that polypeptides expressed by yellow feverrecombinants are processed and presented in a MHC class I-restrictedmanner. In addition, western blotting using antibodies directed againstviral proteins indicated that all yellow fever virus proteins arecorrectly produced and processed in YF-pOva-8 infected cells suggestingthat the foreign antigen was appropriately cleaved away from the viralpolyprotein.

Example 3 Induction of Ova-specific CD8+ T-cells by RecombinantYF-pOva-8

Materials and Methods

CD8+ T cell responses. Immunizations. Groups of 3 C57BL/6 mice were mockinfected or immunized i.v. with 10 7 pfu/mouse of either YFV 17D orrecombinant virus YF-pOva-8. All groups were boosted i.p. with the samedose at day 21 post infection. Seven days later, all mice weresacrificed and their spleens removed and dispersed to single cellsuspensions.

Restimulations and tetramer binding assays. 3×10⁶ splenocytes wererestimulated by co-culturing them for 4 days with 10⁵SIINFEKL-expressing EL4 cells (EL4-SL8) irradiated at 6000 rads.SIINFEKL-MHC class I tetramers were the kind gift of Dr. John Altman(Emory University, Atlanta, Ga.). At day 4, cells were stained asdescribed by Altman et al (23) and analyzed by flow cytometry.

Immunizations and Tumor Challenge. C57BL/6 mice (H-2^(b)) were purchasedfrom the Jackson Laboratory and used between 6-8 weeks of age. Groups of5 or 10 mice were immunized intraperitoneally (i.p.), subcutaneously(s.c.), intramuscularly (i.m.) or intravenously (i.v.) with 3×10⁵pfu/mouse of YF 17D or YF-pOva-8 (Ova-expressing 17D recombinant virus).All groups were boosted with the same dose two weeks later.Non-immunized mice were used as naive controls. Melanoma cells wereharvested by incubation in Ca⁺⁺, Mg⁺ free PBS for 5 minutes, and viablecells counted by trypan blue exclusion and 30 days post infection allmice were challenged with a s.c. injection of 5×10⁴ B16-OVA or B16F0melanoma cells. The size of tumors was determined twice a week andexpressed as tumor area corresponding to the largest perpendiculardiameter in cm². Animals that developed tumors greater than 2.0 weresacrificed.

Immunotherapy of solid tumors. Mice were injected s.c. with 5×10⁴B16-OVA cells. Treatment was started at day 0, 5 or 10 post tumorimplantation and consisted of three s.c. injections of 4×10⁵ pfuYF-pOVA-8 or 17D given in 5 day intervals. A control group was leftuntreated. Mice were observed for tumor development every 3 days andtumors larger than 0.3 cm² were scored as positive.

Immunotherapy of experimental pulmonary metastasis. Mice were injectedi.v. with either 5×10⁴ or 1×10⁵ B16-OVA cells. Immunotherapy wasperformed as described for solid tumors. On day 30, ten mice of eachgroup were sacrificed, then the lungs were removed, placed for 5 minutesin 3% H₂O₂ in H₂O, and then fixed in Bouin's solution (Sigmadiagnostics, St. Louis, Mo., U.S.A.). The H₂O₂ treatment facilitates theanalysis of metastasis under the dissecting microscope by inflating thelungs and bleaching hemorrhages which otherwise could be mistaken formetastases.

Results

To determine whether recombinant yellow fever viruses are able to inducespecific CD8⁺ T-lymphocytes, mice were inoculated with either YF-pOva-8,parental YF-17D, or PBS (naive). Splenocytes obtained from immunizedmice were monitored for the development of a CD8⁻ T-lymphocytepopulation that bound MHC class I-SIINFEKL tetramers (FIGS. 3A-3C).Splenocytes from naive mice, or mice immunized with YF-17D, failed toproduce SIINFEKL specific T-cells. Both freshly isolated splenocytes andin vitro restimulated splenocytes from YF-pOva-8 immunized mice wereassessed for tetrameric binding. Freshly isolated lymphocytes showedminimal tetramer binding after one inoculation (0.5 % of CD8⁻ T-cells).However, after in vitro stimulation a significant percentage of CD8⁺T-cells (8.75%) obtained from mice immunized with YF-pOva-8 werespecific for SIINFEKL.

Protective immunity in vivo. To evaluate whether the vector inducesprotective CTL immunity, we used an established tumor model in whichCTLs play an essential role in protecting the host from challenge with alethal dose of malignant melanoma cells (1). Mice were immunized twicewith YF-pOva-8 or YF-17D and then challenged thirty days later with oneof two C57BL/6 derived melanomas: the parental B16-F0 tumor cell line,or B16-Ova Which stably expresses chicken ovalbumin. Subcutaneousinoculation of naive mice with B16-Ova cells or parental B16-F0 melanomacells yielded tumors that grew with similar kinetics and killed naiveanimals in a few weeks (FIGS. 4A-4D and 5A-5D, data only shown forB16-Ova). Immunization with YF-pOva-8 protected animals against aB16-Ova challenge with a dose 10 times the LD₅₀. Immunization protectedmice from local tumor growth (FIGS. 4A-4D) and also from death (FIGS.5A-D).

We administered the recombinant YF-pOva-8 by four differentroutes—subcutaneous (s.c.), intramuscular (i.m.), intraperitoneal (i.p.)and intraveneous (i.v.)—to compare the efficiency of the protectiveimmune response. Subcutaneous and intravenous inoculation elicitedpotent responses. All of the animals vaccinated with YF-pOva-8 wereprotected at the time when 100% of the control mice had died.Intraperitoneal and intramuscular inoculations were slightly lessefficient; in these groups 10 to 20% of the mice developed tumors anddied. The vaccine effect was specific for SIINFEKL because micevaccinated with YF-pOva-8 were not protected against challenge withparental B16 melanoma cells, which do not express Ova (data not shown).However, we observed a slight, although not significant, delay in tumorgrowth in mice inoculated with YF-17D when the virus was administeredintraperitoneally, subcutaneously or intramuscularly. This effect may bedue to increased cytokine production or other immunological responsesinduced by yellow fever replication. This is not inconsistent with theliterature since it has been shown previously, that IFN-γ and IFN-α haveanti-tumor and anti-cellular activities on B16 melanoma cells (28-30).

Active immunotherapy of established tumors. Next, we determined whethervaccination with YF recombinant is able to induce regression ofestablished tumors. Mice were inoculated subcutaneously with either5×10³ or 5×10⁴ B16-Ova tumor cells and subsequently infected withYF-pOva-8 at the day of tumor implantation (day 0), five days post tumorimplantation (day 5) or ten days post tumor implantation (day 10). Miceinoculated with the lower number of B 16-Ova tumor cells (5×10³),produced tumors in about 60% of unvaccinated mice and were completelyprotected by vaccination, even if treatment was started 10 days aftertumor implantation (FIGS. 6A-6F). For animals inoculated with the higherdoses of tumor cells (5×10⁴), eighty percent of the animals vaccinatedwith YF-pOva-8 at day 0 remained tumor-free 45 days after tumorinjection, while those injected with parental YF-17D or saline developedtumors and died within 3-4 weeks (FIGS. 6A-6F). Vaccination with YF-17Dslightly delayed tumor growth relative to saline, but the effect wasminimal and 90% of the animals developed tumors 30 days after tumorimplantation. Vaccination with recombinant YF-pOva-8 five dayspost-tumor implantation resulted in a partial protection (60% of themice remained tumor-free). Vaccination at day ten had little or noeffect on tumor growth. These results demonstrate that treatment ofestablished tumors can be achieved by vaccination with yellow feverrecombinants.

Active immunotherapy of pulmonary metastasis. B16 melanoma cells, wheninjected into the tail vein of syngenic mice, reproducibly metastasizeto the lungs (31). This provides a model to evaluate whether yellowfever recombinants are able to elicit effective anti-metastaticresponses. Mice were inoculated intravenously with either 5×10⁴ or 1×10⁵B16-Ova cells, and at day 0, 5 or 10 post-tumor inoculation animals werevaccinated subcutaneously with YF-pOva-8 or control virus. Treatmentwith the vector prevented death (FIGS. 9A-9B) and substantially reducedboth the size and number of lung metastasis (FIGS. 7 and 8). Ten weeksafter tumor implantation of 5×10⁴ cells, 100% of the animals vaccinatedat day 0 were healthy (FIGS. 9A-9B). Protection dropped to 80% whenvaccination was started at day 5, and dropped further to 20 % whenanimals were vaccinated starting at day 10 (data not shown). Inoculationwith YF- I 7D had no protective effect, and metastases developed withthe same kinetics as in animals inoculated with saline (FIGS. 7 and 8).When mice were inoculated with 1×10⁵ tumor cells, only 40% of micetreated at day 0 were protected. These results underline the importanceof starting immunotherapy when the tumor burden is low. Nonetheless,these results indicate that recombinant yellow fever viruses expressinga single antigenic epitope are able to elicit a therapeutic anti-tumorresponse in mice.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true-spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

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4 1 8 PRT Artificial Sequence exogenous peptide 1 Ser Ile Ile Asn PheGlu Lys Leu 1 5 2 55 DNA Artificial Sequence synthesized oligonucleotide2 atcgcggacc gagtggtttt gtgtttgtca tccaaaggtc tgcttattct tgagc 55 3 24DNA Artificial Sequence synthesized oligonucleotide 3 caatgaggcactcgcagcag ctgg 24 4 23 DNA Artificial Sequence synthesizedoligonucleotide 4 tgccctagct ctgtgcgctg ccc 23

What is claimed is:
 1. An isolated replication-competent recombinantyellow fever virus comprising an insertion of an exogenous nucleic acidsequence encoding an exogenous polypeptide, wherein infection of a hostwith the recombinant virus provides for expression of the exogenouspolypeptide by the host cell and induction of an immune response in ahost.
 2. The isolated recombinant yellow fever virus of claim 1, whereinthe exogenous nucleic acid sequence is expressed as a component of arecombinant viral polyprotein precursor.
 3. The isolated recombinantyellow fever virus of claim 2, wherein the recombinant polyproteinprecursor comprises a proteolytic cleavage site.
 4. The isolatedrecombinant yellow fever virus of claim 3, wherein the proteolyticcleavage site is positioned to provide for release of the exogenouspolypeptide upon proteolytic processing.
 5. The isolated recombinantyellow fever virus of claim 1, wherein the exogenous polypeptide is apolypeptide of a virus other than yellow fever virus.
 6. The isolatedrecombinant yellow fever virus of claim 1, wherein the exogenouspolypeptide is a polypeptide of a microbial pathogen other than yellowfever virus.
 7. The isolated recombinant yellow fever virus of claim 1,wherein the virus is live and attenuated.
 8. The isolated recombinantyellow fever virus of claim 1, wherein the exogenous polypeptide isexpressed on the surface of the host cell following infection.
 9. Theisolated recombinant yellow fever virus of claim 1, wherein theexogenous polypeptide is secreted from the host cell followinginfection.
 10. An isolated host cell infected with the recombinantyellow fever virus of claim
 1. 11. A composition comprising therecombinant yellow fever virus of claim 1, and a buffer.
 12. Thecomposition of claim 11, further comprising a pharmaceuticallyacceptable excipient.
 13. A method of eliciting an immune response in amammalian host to an antigenic polypeptide, comprising: administering arecombinant yellow fever virus of claim 1 to a mammalian host, whereinthe exogenous polypeptide is an antigenic polypeptide, wherein saidadministering provides for infection of a host cell and expression ofthe antigenic polypeptide; wherein expression of the exogenouspolypeptide results in induction of an immune response in the host tothe antigenic polypeptide.
 14. The method of claim 13, wherein theantigenic polypeptide is a polypeptide of a microbial pathogen.
 15. Anisolated replication-competent recombinant yellow fever virus comprisinga sequence encoding a recombinant polyprotein precursor, the polyproteinprecursor comprising an insertion of an exogenous nucleic acid sequenceencoding an exogenous polypeptide.
 16. The isolated recombinant yellowfever virus of claim 15, wherein the polyprotein precursor comprises aproteolytic cleavage site such that the exogenous polypeptide isreleased from the recombinant polyprotein precursor upon proteolyticprocessing.
 17. The isolated recombinant yellow fever virus of claim 15,wherein the exogenous polypeptide is a polypeptide of a virus other thanyellow fever virus.
 18. The isolated recombinant yellow fever virus ofclaim 15, wherein the virus is live and attenuated.
 19. The isolatedrecombinant yellow fever virus of claim 15, wherein the exogenouspolypeptide is expressed on the surface of the host cell followinginfection.
 20. The isolated recombinant yellow fever virus of claim 15,wherein the exogenous polypeptide is secreted from the host cellfollowing infection.
 21. An isolated host cell infected with therecombinant yellow fever virus of claim
 15. 22. A composition comprisingthe recombinant yellow fever virus of claim 15, and a buffer.
 23. Thecomposition of claim 22, further comprising a pharmaceuticallyacceptable excipient.